Method for carrying out organic chemical reactions and appartaus for carrying out that method

ABSTRACT

In carrying out an organic chemical reaction, such as the synthesis of a polypetide, it is known to attach a substance to particles of pervious support material and to immerse the particles in a sequence of liquids which serve as reagents, solvents etc. The invention provides a method in which, during at least one stage in a reaction of that kind, a liquid is introduced into a vessel containing particles of support material in such a manner that the immediately preceding liquid is progressively displaced by the incoming liquid. Apparatus for carrying out that method comprises a vessel (1) with upper and lower sintered restraining plates (5 and 6) between which the support material is located. Valves (8 and 11) can be set at will to enable a pump (13) to pump liquid into the vessel from above or below and to recirculate or discharge the liquid.

This invention relates to a method of carrying out organic chemicalreactions and apparatus for carrying out that method.

The invention has largely been developed for use in carrying out thesynthesis of peptides but it will be understood that it is of broaderapplication than that and can be used in carrying out a wide range ofother organic chemical reactions.

In a known type of procedure, one of the substances that takes part in areaction is attached to particles of a pervious support material so thatat least some of the substance is within the particles. The material maybe relatively rigid but it is preferred to use a soft gel. In carryingout the reaction, support material to which the substance is attachedhas a sequence of liquids applied to it, one after another. Some of theliquids are reagents which are used to bring about steps in the reactionprocess, while other liquids are solvents which are used for the washingor other treatment of the support material or of the substance attachedto it. Liquids serving other purposes may also be employed. It is usualto place a quantity of the support material in a vessel which is open atthe top and which has an outlet at the bottom leading to a dischargetube. A tap is provided adjacent to the outlet to enable the outlet tobe opened and closed at will. Downstream of the tap a branch extendslaterally from the discharge tube and can be connected to a vacuum pump.Inside the vessel, above the outlet, is a transverse plate make ofsintered material; liquid can pass through the pores in the plate butthe pores are too small to permit the particulate support material topass through them.

In carrying out the method a first liquid is introduced into the vesselso that the support material becomes immersed in it. The vessel isshaken to allow the liquid to come into intimate contact with theparticulate support material After a predetermined period of time haselapsed the tap is opened and the first liquid is drained off throughthe discharge tube; a vacuum is applied to the branch and assists thewithdrawal of the liquid from the vessel. When this occurs, some of theliquid tends to remain trapped within the particles, and the particlesthemselves tend to pack together. In particular, if the particles aresoft rather than rigid they tend to pack together to form asubstantially impervious bed.

After as much as possible of the first liquid has been withdrawn, asecond liquid is introduced into the vessel and the vessel is shakenagain to allow the second liquid to come into contact with the supportmaterial. After a further predetermined period of time has elapsed thetap is re-opened and the second liquid is drained off in the same waythat the first liquid was drained off. The process is then repeated withas many liquids as are required to complete the desired reaction orreactions.

As it is impossible to withdraw any of the liquids fully from thesupport material, each newly introduced liquid tends to be slightlycontaminated with the previous liquid. This may lead to a reduced yieldor to the need for the addition of extra steps in each of which theremaining unwanted liquid is washed by the introduction of a suitableliquid. An aim of the present invention is to enable those problems tobe overcome or at least reduced.

From one aspect the present invention consists in a method of carryingout an organic chemical reaction in which a substance which takes partin the reaction is attached to particles of previous support material sothat at least some of the substance is within the particles, a pluralityof liquids is successively introduced into a vessel containing aquantity of that support material so that the material becomes immersedin each of those liquids successively and the reaction occurs as aconsequence of the immersion of the quantity of support material in thatsuccession of liquids, the introduction of at least one of the liquidsinto the vessel being carried out in such a manner that the immediatelypreceding liquid is progressively displaced by the incoming liquid,these two liquids being of different densities and the arrangement beingsuch that, during the introduction of the incoming liquid into thevessel, the more dense of said two liquids is situated below the lessdense of said two liquids.

An important feature of the present invention is the progressivedisplacement of one liquid by the next. As this occurs there is normallya reasonably well-defined boundary or interface between the two liquids.This progressive displacement is also referred to herein as layereddisplacement. An advantage of this progressive or layered displacementis that the incoming liquid tends to wash the previous liquid frominside the particles, so that the tendency for the one liquid to becontaminated by the preceding liquid is reduced.

When the incoming liquid is more dense than the liquid that immediatelyprecedes it in the vessel, the incoming liquid is preferably introducedinto the vessel from below. This helps to maintain the integrity of thetwo liquids and to prevent their mixing together For the same reason,when the incoming liquid is less dense than the liquid that immediatelyprecedes it in the vessel, the incoming liquid is preferably introducedinto the vessel from above.

The support material is normally such that when a particle of thematerial has been immersed in a liquid for a while and reaches anequilibrium with the liquid much of the overall volume of the particleis occupied by the liquid. Consequently, the density of theliquid-filled particle is substantially equal to the density of theliquid itself.

When particles of the support material are immersed in one liquid whichis then progressively displaced by another liquid of a differentdensity, as described above, the particles tend to remain in the oneliquid and to move with that liquid, ahead of the incoming liquid. In apreferred method of carrying out the invention said one liquid, as it isdisplaced, passes through porous restraining means of a pore-size suchthat the support material cannot pass through it. As the incoming liquidprogressively displaces the immediately preceding liquid the particlesof support material tend to move towards the restraining means.Eventually the leading particles are restrained from further movement bythe presence of the restraining means, the following particles packagainst them, and this continues until a bed of packed particles ofsupport material is formed against the restraining means. The incomingliquid then passes progressively through the bed and tends to displacethe immediately preceding liquid from inside the particles of supportmaterial. This is found to be a particularly efficient method ofreplacing one liquid with another inside the particles.

Provided that the densities of the two successive liquids aresignificantly different a stable, coherent bed of support material isformed If the difference in density is very small, however, the bed maybe less stable and there may be some slight movement of the constituentparticles in the bed. Such a bed is referred to herein as a semi-packedbed. A method in accordance with the invention is nevertheless found tooperate satisfactorily with a semi-packed bed.

When the incoming liquid has displaced the immediately preceding liquidfrom the support material, the overall density of each particle becomesvery close to that of the incoming liquid As a result the forces actingon the particles to maintain them in a packed state are reduced, and atleast some of the particles may become free from the bed. This does notaffect the method adversely, however, as the particles concerned arethose in which the incoming liquid has replaced the immediatelypreceding liquid.

In order to ensure that the bed of support material is fully broken up,the incoming liquid is preferably recirculated before it in turn isreplaced, the recirculation including flow through the restraining meansin a reverse direction. The recirculation is preferably continuous orsubstantially so and may be such as to cause the particles of supportmaterial to separate from one another and to move randomly around in theliquid in the vessel. The support material is then in a state referredto herein as fluidised.

It is generally desirable to cause the support material to becomefluidised on each occasion after it has been formed into a bed andincoming liquid has been progressively passed through it. Fluidisationenables the new liquid to come into intimate contact with each particleof the support material individually. Also, where soft-gel supportmaterial is used, fluidisation enables each particle to achieveequilibrium with the liquid and, without restraint, to swell or shrinkto a size dependent on the nature of the liquid. When the particles aresubsequently formed into a new bed, there is then no tendency forover-tight packing to occur or for cracks or fissures to form in thebed, both of which phenomena would be disadvantageous as they wouldmilitate against the steady flow of the incoming liquid through theparticles in the bed.

The bed or beds of particulate support material referred to above may beformed at or near the bottom of the vessel or at or near the top of thevessel, depending on the relative densities of the particles of supportmaterial and of the liquid in which they are immersed. It may thereforehappen that a bed of particulate support material is immersed in aliquid of a relatively low specific gravity so that it resides at ornear the bottom of the vessel and that as the next succeeding liquid isintroduced into the vessel from below, being a liquid with a relativelyhigh specific gravity, the bed rises as a whole to a position at or nearthe top of the vessel. Conversely a bed of support material supported ator near the top of the vessel in a relatively dense liquid may movedownwards as a whole when a less dense liquid is introduced into thevessel from above. In general, however, it is preferred to break up thebed by recirculation before a new bed is formed.

It will be appreciated from the foregoing that during the progressivedisplacement of one liquid by another the direction of flow may beupwards or downwards, depending on the relative densities of theliquids. Apparatus for use in carrying out the invention may thereforeemploy upper and lower restraining means so that beds of supportmaterial can be formed against one or the other, depending on thedirection of flow of the incoming liquid There are preferably two suchrestraining means spaced apart, one above the other, and definingbetween them an enclosure in which the support material is trapped. Therestraining means or each restraining means preferably comprises a plateof a sintered material. Preferably the incoming liquid is introducedthrough one of the restraining means while the immediately precedingliquid escapes through the other restraining means. The incoming liquidpreferably flows in through the whole or substantially the whole of therestraining means; this encourages uniform displacement of theimmediately preceding liquid. Nevertheless it has been observed thateven if part of the surface of a restraining means is blocked, theincoming liquid, after passing through the unblocked part of therestraining means normally spreads out over the whole of the surface ofthe restraining means, provided that the incoming liquid is introducedat a sufficiently slow rate.

While the invention includes within its scope a method in which thesupport material is immersed successively in only two liquids, it isenvisaged that the invention will usually be employed in carrying outmethods in which the support material is immersed in each in turn of aconsiderably greater number of liquids. Nevertheless it is also to beunderstood that one or more of the liquids may be introduced in a mannerdifferent from the manner that is characteristic of the presentinvention and in which the immediately preceding liquid is progressivelydisplaced by the incoming liquid For example, it may be necessary ordesirable for the support material at some stage to be immersed in oneliquid and for it next to be immersed in a mixture of that liquid withanother liquid. To achieve this result, some of that other liquid may beintroduced into the vessel in such a manner as to mix with theimmediately preceding liquid rather than to displace it completely.

While the invention can be used with support material comprisingparticles of any suitable form, including rigid particles such as thosemade from polystyrene, it is particularly suitable for use with asupport material comprising a soft gel in particulate form. The use ofsuch gels in carrying out organic chemical reactions is alreadywell-known and well-understood, so the gels will not be furtherdescribed here. As is already known, when such gels in particulate formare immersed in a liquid they absorb some of the liquid and swell to asize dependant on the nature of the liquid. When immersed in asuccession of different liquids, the particles tend to change in size.When using a method in accordance with the present invention, in whichthe particles remain immersed, the particles can readily be caused todisperse temporarily and move relative to one another in order toaccommodate the changes in size before being reformed into a new bed.

From another aspect the present invention consists in apparatus for usein carrying out a method according to the first aspect of the presentinvention and comprising a vessel for containing a quantity of supportmaterial in particulate form, the vessel having upper and lowerpermeable restraining means through which liquids can be introduced intoand withdrawn from the vessel, and pumping means operative to pumpliquid into the vessel from above and also operative to pump liquid intothe vessel from below.

The apparatus preferably incorporates valve means which in one stateenables liquid to be pumped into the vessel from above and in anotherstate enables liquid to be pumped into the vessel from below. The valvemeans is preferably such that in said one state it guides liquiddisplaced from below to enter a return duct, and in said other state itguides liquid displaced from above to enter the return duct. There mayalso be subsidiary valve means which in one state connects the returnduct to the inlet of the pump, and in another state connects a liquidsupply tank to the inlet of the pump and connects the return duct to adischarge duct.

An embodiment of the present invention will now be described, by way ofexample only, with reference to the accompanying drawings, in which:

FIG. 1 is a diagrammatic view of apparatus embodying the presentinvention, and

FIG. 2 is a graph of a chromatogram.

The apparatus illustrated is largely assembled from conventionalcomponents. The details of those components have no real bearing on thepresent invention and therefore the apparatus is shown in largelydiagrammatic form.

The apparatus comprises a vessel 1 having a cylindrical wall 2, with itsaxis vertical, and frusto-conical end pieces 3 and 4 at the top andbottom respectively The lower end piece 4 is fixed relative to the wall2 but the upper end piece 3 can be adjusted vertically so as to vary theinternal volume of the vessel. A restraining plate 5 is mounted acrossthe mouth of the upper end piece 3 and a similar plate 6 is mountedacross the mouth of the lower end piece 4. The plates 5 and 6 arehorizontal and each is made from sintered PTFE.

The upper end of the upper end piece 3 is connected by a tube 7 to afirst port of a valve 8 which is a two-way, two-position rotary valve.The lower end of the lower end piece 4 is connected by a tube 9 to asecond port of the valve 8, the first and second ports being opposed toeach other so that in no position of the valve are they interconnected.A third port of the valve 8 is connected by means of a tube 10 to afirst port of a valve 11 which is similar in construction and operationto the valve 8. The fourth port of the valve 8 is connected to one endof a length of flexible tubing 12 constituting part of a peristalticpump 13. The tubing 12 is of arcuate form, being mounted between anarcuate abutment 14 and a rotor 15 which is provided with rollers (notshown) which engage the length of tubing 12 in the usual manner. Therotor is connected to a reversible motor (not shown) which is ofvariable speed and causes the rotor to rotate in the direction of thearrow 16. The other end of the length of tubing 12 is connected to asecond port of the valve 11. A third port of the valve 11 is connectedto the outlet of a reservoir 17, while the fourth port of the valve 11is connected to a discharge tube 18.

The other end of the length of tubing 12 is made of a chemically inertmaterial, a suitable material being that marketed under the trade markMarprene. The other tubes 7, 9, 10 and 18 are also made from achemically inert material, a suitable material being PTFE.

In use, a quantity of pervious support material (not shown) inparticulate form is disposed in the vessel 1 between the upper and lowerrestraining plates 5 and 6. Preferably the material occupies more thanhalf of the available volume, the material typically occupying about twothirds of that volume.

In a typical operation of the apparatus a first liquid is introducedinto the reservoir 17. The valve 11 is turned to a first position(position I), in which the outlet of the reservoir is connected to theinlet of the pump 13, and the valve 8 is also turned to a first position(position I), in which the outlet of the pump 13 is connected to thetube 9. Both valves are illustrated as being in position I. The pump isoperated to pump the first liquid from the reservoir 17 to the lower endpiece 4 of the vessel 1. Excess liquid passes from the top of the vesselthrough the tube 7 to the valve 8 and thence through the tube 10 to thevalve 11 and the discharge tube 18. When this occurs the valve 11 isturned to a second position (position II) such that the tube 10 isconnected to the inlet of the pump 13 while the reservoir 17 isconnected to the discharge tube 18. Continued operation of the pumpcauses the liquid to be recirculated through the vessel, while anyremaining contents of the reservoir 17 are drained away through thedischarge tube 18. The rate of operation of the pump is such that theparticles of support material are in a fluidised state.

A second liquid is introduced into the reservoir and the valve 11 isreturned to position I so that the second liquid is drawn through thepump 13. If, for the sake of example, the second liquid is more densethan the first liquid, the valve 8 is allowed to remain in position Iand the second liquid is progressively introduced into the vesselthrough the tube 9. The rate of operation of the pump is such that theincoming second liquid progressively displaces the first liquid, theboundary between the two liquids moving steadily upwards. Theparticulate support material forms a bed against the upper restrainingplate 5 as described above. When all of the first liquid has beendischarged from the vessel 1 and the second liquid has reached the valve11, the valve 11 is turned back to position II, while the valve 8 isturned to a second position (position II) in which the outlet of thepump 13 leads to the tube 7 while the tube 9 is connected to the tube10. Consequently the second liquid is recirculated through the vessel ina direction the reverse of that in which it was first introduced intothe vessel. This breaks up the bed and fluidises the support materialwhich then circulates freely within the confines of the vessel betweenthe restraining plates 5 and 6. It may well have happened that duringtheir immersion in the second liquid the particles have changed involume, for the extent to which each particle swells when immersed in aliquid often varies from liquid to liquid. If the particles had not beencaused to circulate freely for a while in the second liquid, theirchange in volume would have tended to lead either to increased packingwithin the bed resulting in an increased resistance to flow of anysubsequent liquid or to the formation of cracks or fissures through thebed resulting in a tendency for any subsequent liquid to flow throughthe cracks or fissures without permeating through the bed as is desired.

A plurality of other liquids can then be introduced, one after another,in much the same manner. When the incoming liquid is less dense than thepreceding liquid it can be introduced into the vessel 1 from above byway of the tube 7, the valve 8 having been rotated to position II.

By suitable variation in the speed of the pump 13 and manipulation ofthe valves 8 and 11 various other effects may be obtained, as indicatedabove. For example, by turning valve 11 from position I to position IIbefore one liquid has been only partially replaced by a subsequentliquid, and if necessary by increasing the speed of the pump, a mixtureof liquids can be formed which is recirculated through the vessel 1.

When a reaction has been completed the support material, bearing theproduct of the reaction, may be withdrawn from the vessel and separatedfrom it by known techniques.

It will be appreciated from the foregoing description of the apparatusillustrated that numerous variations in technique are possible Incarrying out any particular chemical reaction, the sequence oftechniques employed is preferably chosen so as to maximise the yield andminimise the contamination of the product with unwanted materials.

There follows an example of a reaction carried out by a method inaccordance with the present invention and using apparatus of the kindillustrated.

EXAMPLE

This reaction consists of the synthesis of a calcitonin gene relatedpeptide segment, CGRP (31-36).

The method employs an acid-catalyzed N-terminal deprotection strategywith t-butoxycarbonyl (Boc) amino groups.

Particulate support material was prepared. This comprises particles ofcrosslinked poly[N-[2-(4-hydroxyphenyl)ethyl]acrylamide] (Copolymer Q).

Copolymer Q, a de-O-acetylated bead form copolymer ofN-[2-(4-hydroxyphenyl)ethyl]acrylamide and N,N'-diacryloylpiperidine(molar ratio 20/1), swollen volume in HCONMe₂ 10-20 cm³ g⁻¹ copolymer,was prepared according to the literature (Epton, R. and Williams, A.Int. J.Biol. Macromol. 1981, 3, 336).

A solution of Boc-Ala-OH (2.37 g, 12.5 mmol) diisopropylcarbodiimide(DIC) (1.90 g, 15 mmol) in HCONMe₂ (15 cm³) was added to Copolymer Q(0.5 g, phenol content 5.0 mmol g⁻¹) 4-dimethylaminopyridine (DMAP)(0.61 g, 5 mmol) was added with just sufficient HCONMe₂ (˜15 cm³) topermit nitrogen stirring. The reaction was allowed to proceed over 18 hat 25° C. with gentle nitrogen stirring Reagent solution was drained offand the gel beads washed repeatedly with HCONMe₂, CH₂ Cl₂ and Et₂ O anddried to give Boc-Ala-O-[Copolymer Q] (0.93 g 2.3 mmol amino acid).

Boc-Lys(ClZ)-OH (1.24 g, 3 mmol) and 1-hydroxybenzotriazole (HOBt) (0.81g, 6 mmol) in HCONMe₂ (7.5 cm³) was chilled to 0° C. anddiisopropylcarbodiimide (DIC) (0.34 g, 2.7 mmol) added. The solution wasstirred gently and then allowed to warm to room temperature over 30 min.N-methylmorpholine (NMM) (0.20 g, 2 mmol) was added immediately prior touse.

Boc-Ser(Bzl)-OBt/HOBt solution was prepared in similar manner toBoc-Lys(ClZ)-OBt/HOBt solution (described in the preceding paragraph)from Boc-Ser(Bzl)-OH (0.88 g, 3 mmol).

Boc-Gly-OBt/HOBt solution was prepared in similar manner toBoc-Lys(ClZ)-OBt/HOBt solution from Boc-Gly-OH (0.43 g, 3 mmol).

Boc-Val-OBt/HOBt solution was prepared in similar manner toBoc-Lys(ClZ)-OBt/HOBt solution from Boc-Val-OH (0.65 g, 3 mmol).

Boc-Asn-OBt/HOBt solution was prepared in similar manner toBoc-Lys(ClZ)-OBt/HOBt solution from Boc-Asn-OH (0.70 g, 3 mmol).

-The vessel of the apparatus, which had a height of 2.5 cm and adiameter of 2.5 cm, was batched with Boc-Ala-O-[Copolymer Q] (0.37 g, 1mmol amino acid). 3-MeC₆ H₄ OH/Cl₃ CMe (4/1) was pumped in to fill thereactor by upward flow, and swelling was allowed to proceed with upwardrecycling over 1 h. The particles of copolymer moved freely in thevessel, this being referred to in the accompanying Table as a fluidisedstate of the bed. The appropriate standard CF peptide chain elongationcycles were then performed (see the Table). Each in turn of the fiveamino acid 1-benzotriazole ester/HOBt solutions described above wasemployed in a cycle of operations with the result that each in turn ofthe amino acid was added to the chain. Each cycle consisted of a prewash(or post-coupling wash), CF₃ CO₂ H/3-MeC₆ H₄ OH/Cl₃ CMe (5/4/1) mediateddeprotection, post-deprotection washing, neutralisation byN-methylmorpholine (NMM) in CHONMe₂, post-neutralisation washing,treatment with an appropriate Boc amino acid 1-benzotriazoleester/HOBt/NMM solution to effect peptide chain elongation and, finally,post-coupling washing. Full operational and experimental details of thestandard CF peptide chain extension cycles, together with details ofreaction and washing solvents, are given in the Table. A non-standard CFpeptide chain extension cycle, in which post-deprotection neutralisationand post-neutralisation washings were omitted (Table: Stages 4 and 5) infavour of relying solely on in situ neutralisation during the laterpeptide bond formation step, is described in Note 1 to the Table. Thisnon-standard cycle was used to extend the peptide chain fromBoc-Lys(ClZ)-Ala-O-[Copolymer Q] toBoc-Ser(Bzl)-Lys(ClZ)-Ala-O-[Copolymer Q].

Upon completion of the synthesis, the solvent-swollen gel assembly wasremoved from the reactor and washed repeatedly with HCONMe₂, CH₂ Cl₂ andEt₂ O and then dried to giveBoc-Asn-Val-Gly-Ser(Bzl)-Lys(ClZ)-Ala-O-[Copolymer Q] (Peptide-resinAssembly 1) (1.12 g).

Peptide-resin Assembly 1 (50 mg) was allowed to swell to equilibrium inHCONMe₂ (1.98 cm³) and 98% NH₂ NH₂.H₂ O (0.02 cm³) added with nitrogenstirring. Reaction was allowed to proceed for 2 min, after which thereaction liquor was drawn into Et₂ O (50 cm³), prechilled at -78° C.,and the precipitate collected and dried to giveBoc-Asn-Val-Gly-Ser(Bzl)-Lys(ClZ)-Ala-NHNH₂ (˜39 mg). An HPLCchromatogram for this compound, performed on a Waters Novapak reversedphase C-18 radial pak cartridge (100×8 mm) with 42% aqueous MeCN aseluent at a flow rate of 1.5 cm³ min⁻¹, is shown in FIG. 2. IN thisgraph the horizontal axis represents time and is calibrated in minutes.

The foregoing example illustrates the use of the invention in peptidesynthesis by a method employing an acid-catalyzed N-terminaldeprotection strategy with Boc amino acids. The invention can equallywell be used in peptide synthesis by a method employing an aminolyticN-terminal deprotection strategy with fluorenylmethoxycarbonyl aminoacids.

                                      TABLE                                       __________________________________________________________________________    Details of operations involved in performing a standard                       1 mmol scale ultra-high load CF chain extension cycle                         with acid-catalysed N-terminal deprotection (Boc amino                        acids).                                                                       __________________________________________________________________________                                   Flow Direc-                                                                   tion in                                        Stage of cycle                                                                             Reagent or solvent                                                                              Vessel                                         __________________________________________________________________________    1. prewash   1a.                                                                              3-MeC.sub.6 H.sub.4 OH/Cl.sub.3 CMe(4/1)                                                     up                                             2. Deprotection                                                                            2a.                                                                              CF.sub.3 CO.sub.2 H                                                                          up                                                          2b.                                                                              CF.sub.3 CO.sub.2 H (5 min)                                                                  down                                                        2c.                                                                              CF.sub.3 CO.sub.2 H/3-MeC.sub.6 H.sub.4 OH/                                                  up                                                             Cl.sub.3 CMe (5/4/1) (25 min)                                 3. Post-deprotec-                                                                          3a.                                                                              3-MeC.sub.6 H.sub.4 OH/Cl.sub.3 CMe (4/1)                                                    down                                              tion wash                                                                               3b.                                                                              3-MeC.sub.6 H.sub.4 OH/Cl.sub.3 CMe (4/1)                                                    up                                                          3c.                                                                              HCONMe.sub.2   down                                                        3d.                                                                              HCONMe.sub.2   up                                             4. Neutralisation                                                                          4a.                                                                              NMM            down                                              (Note 1)                                                                                4b.                                                                              NMM/HCONMe.sub.2 (1/9)                                                                       up                                             5. Post-neutralis-                                                                         5a.                                                                              HCONMe.sub.2   up                                                ation wash (Note 1)                                                                     5b.                                                                              HCONMe.sub.2   down                                           6. Coupling  6a.                                                                              Boc--AA--OBt/HoBt/NMM                                                                        up                                                             HCONMe.sub.2                                                               6b.                                                                              Boc--AA--OBt/HoBt/NMM                                                                        up                                                             HCONMe.sub. 2 (60 min) (Note 2)                               7. Post-coupling                                                                           7a.                                                                              HCONMe.sub.2   down                                              wash                                                                                    7b.                                                                              HCONMe.sub.2   up                                                          7c.                                                                              3-MeC.sub.6 H.sub.4 OH/Cl.sub.3 CMe                                                          up                                                             (4/1)                                                                      7d.                                                                              3-MeC.sub.6 H.sub.4 OH/Cl.sub.3 CMe                                                          down                                                           (4/1)                                                         __________________________________________________________________________    Stage   Operation   Delivery mode                                             __________________________________________________________________________    1a.     Washing     Uniform                                                   2a.     Dispensing  Uniform (layered displace-                                                    ment)                                                     2b.     Back cycle/ Mixing                                                            deprotection                                                          2c.     Deprotection                                                                              Uniform circulation                                       3a.     Washing     Uniform (layered displace-                                                    ment)                                                     3b.     Washing     Uniform                                                   3c.     Washing     Uniform (layered displace-                                                    ment)                                                     3d.     Washing     Uniform                                                   4a.     Dispensing  Uniform (layered displace-                                                    ment)                                                     4b.     Back cycle/ Mixing/Uniform circulation                                        neutralisation                                                        5a.     Washing     Uniform (layered displace-                                                    ment)                                                     5b.     Washing     Uniform                                                   6a.     Dispensing  Uniform (layered displace-                                                    ment)                                                     6b.     Recycle to  Uniform                                                           couple                                                                7a.     Washing     Uniform (layered displace-                                                    ment)                                                     7b.     Washing     Uniform                                                   7c.     Washing     Uniform (layered displace-                                                    ment)                                                     7d.     Washing     Uniform                                                   __________________________________________________________________________                    Reagent or                                                                    solvent Position                                                                             Position                                             State of bed of                                                                         used or of value                                                                             of value                                       Stage support material                                                                        added   8      11                                             __________________________________________________________________________    1a.   Fluidised 30 cm.sub.3                                                                           I      I                                              2a.   Packed at top                                                                           10 cm.sup.3                                                                           I      I                                              2b.   Fluidised none    II     II                                             2c.   Fluidised none    I      II                                             3a.   Packed at 30 cm.sup.3                                                                           II     I                                                    bottom                                                                  3b.   Fluidised 30 cm.sup.3                                                                           I      I                                              3c.   Packed at 30 cm.sup.3                                                                           II     I                                                    bottom                                                                  3d.   Fluidised 30 cm.sup.3                                                                           I      I                                              4a.   Semi-packed                                                                             2.0 cm.sup.3                                                                          II     I                                                    towards bottom                                                          4b.   Fluidised none    I      II                                             5a.   Fluidised 30 cm.sup.3                                                                           I      I                                              5b.   Packed at 30 cm.sup.3                                                                           II     I                                                    bottom                                                                  6a.   Semi-packed                                                                             7.5. cm.sup.3                                                                         I      I                                                    towards top                                                             6b.   Fluidised none    I      II                                             7a.   Packed    30 cm.sup.3                                                                           II     I                                              7b.   Fluidised 30 cm.sup.3                                                                           I      I                                              7c.   Packed    30 cm.sup.3                                                                           I      I                                              7d.   Packed    30 cm.sup.3                                                                           II     I                                              __________________________________________________________________________     Note 1. On progressing peptide chain elongation from the dipeptide to         tripeptide stage, a nonstandard chain elongation cycle was performed in       which postdeprotection neutralisation and postneutralisation washings wer     omitted (Stages 4 and 5). This was necessary to precent the free              aminoterminal of the dipeptide attacking the phenyl ester anchoring           linkage and causing deloading via diketopiperazine formation.                 Note 2. For Boc-- Gly--OBt coupling time was 90 min.                     

We claim:
 1. A method of carrying out an organic chemical reaction inwhich a substance which takes part in the reaction is attached toparticles of pervious support material so that at least some of thesubstance is within the particles, a plurality of liquids issuccessively introduced into a vessel containing a quantity of thatsupport material so that the material becomes immersed in each of thoseliquids successively and the reaction occurs as a consequence of theimmersion of the quantity of support material in that succession ofliquids, the method being characterised in that the introduction of atleast one of the liquids into the vessel is carried out in such a mannerthat the immediately preceding liquid is progressively displaced by theincoming liquid, these two liquids being of different densities and thearrangement being such that, during the introduction of the incomingliquid into the vessel, the more dense of said two liquids is situatedbelow the less dense of said two liquids.
 2. A method according to claim1 characterised in that the pervious support material comprises a softgel in particulate form.
 3. A method according to claim 1 characterisedin that said incoming liquid is more dense than said immediatelypreceding liquid and is introduced into the vessel from below.
 4. Amethod according to claim 1 characterised in that said incoming liquidis less dense than said immediately preceding liquid and is introducedinto the vessel from above.
 5. A method according to claim 3characterised in that said immediately preceding liquid, as it isdisplaced, passes through porous restraining means of a pore-size suchthat the support material cannot pass through it.
 6. A method accordingto claim 2 characterised in that at least one of said liquids isrecirculated after its initial introduction into the vessel, thedirection of flow being the reverse of that in which the liquid wasinitially introduced into the vessel.
 7. A method according to claim 2characterised in that said incoming liquid is introduced into the vesselthrough a porous restraining means having pores of too small a size toallow the particles of support material to pass through them. 8.Apparatus for use in carrying out a method according to claim 1characterised in that it comprises a vessel for containing a quantity ofsupport material in particulate form, the vessel having upper and lowerpermeable restraining means through which liquids can be introduced intoand withdrawn from the vessel, a pumping means operative to pump liquidinto the vessel from above and also operative to pump liquid into thevessel from below, and a first valve means, wherein said valve means hasa first and second state and in one of said states connects a returnduct to an inlet of said pump and in the second of said states connectsa liquid supply tank to said inlet of said pump and connects the returnduct to a discharge duct.
 9. Apparatus according to claim 8characterised in that it incorporates a second valve means (8) which inone state enables liquid to be pumped into the vessel from above and inanother state enables liquid to be pumped into the vessel from below.10. Apparatus according to claim 9 characterised in that the valve means(8) is such that in said one state it guides liquid displaced from belowto enter a return duct (10), and in said other state it guides liquiddisplaced from above to enter the return duct.
 11. Apparatus accordingto claim 8 characterised in that the pump is a peristaltic pump.